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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
  • C12N9/1088—Glutathione transferase (2.5.1.18)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates to vaccines for the preventative treatment for infection of liver fluke in ruminant animals.
• the invention also relates to methods for the preventative treatment for infection of liver fluke in ruminant animals.
• Fascioliasis Effective control of infection with liver fluke (Fascioliasis) is a major worldwide problem in the animal industry. Fascioliasis is caused by infection with the trematode parasite Fasciola hepatica (F. hepatica). In particular, in ruminants such as sheep and cattle, it can cause serious economic losses due to wasting, death and reduced wool and milk production [1]. Current control methods rely heavily on the use of anthelmintic chemicals but these methods are not always effective [2].
• GST glutathione-S- transferase
• EC 2.5.1.18 are a family of multifunctional proteins involved in the metabolism of a broad range of xenobiotics and the binding and possible transport of endogenous anionic compounds such as bilirubin and heme [9].
• electrophilic substrates are neutralised following conjugation with glutathione, rendering the product water soluble and facilitating excretion.
• US Patent Specification 4743446 (National Research Development Corp) describes antigens specific to the juvenile stage of F. hepatica which are prepared by raising an antiserum against the juvenile flukes, absorbing this antiserum with antigens extracted from adult flukes, separating the immunoglobulins (Ig) from the unabsorbed antiserum and using these Ig to affinity purify juvenile-specific antigens (JSA) from lysates of juvenile fluke.
• JSA juvenile-specific antigens
• European Patent Specification 11438 (Vaccines International Ltd) describes a vaccine against bovine fascioliasis comprising irradiated metacercariae of F. ⁇ i ⁇ antica. The use of irradiated metacercariae for vaccination of sheep a ⁇ ainst F. hepatica has been reported to be unsuccessful [12].
• the present invention provides in one form a vaccine for the preventative treatment for infection of liver fluke in ruminants, the vaccine comprising glutathione-S-transferase (GST) derived from adult worms of F. hepatica.
• GST glutathione-S-transferase
• a vaccine containing GST is able to stimulate immunity in sheep to infection with metacercariae of F. hepatica.
• the GST proteins are purified from adult worms of F. hepatica by affinity chromatography on glutathione-agarose.
• the GST proteins purified by glutathione-agarose chromatography comprise a mixture of proteins of similar molecular weight of about 26,000 and 26,500 Da. These proteins can be fractionated by two dimensional SDS-PAGE into about 10-11 individual components with different apparent pi values.
• the GST used in the present invention may be extracted as described above or alternatively the parts of the molecule responsible for this vaccination effect may be synthesised as peptide molecules or by means of genetic engineering. It will be appreciated that a protective immune response can be achieved by vaccination with a peptide fragment of the GST described. Anti-idiotype antibodies corresponding to the vaccinating epitopes of the GST molecule may also be used as a vaccine.
• Fasciola ⁇ i ⁇ antica which is believed to be the predominant cause of liver fluke infection in tropical zones.
• the vaccine further comprises adjuvants.
• adjuvants Any adjuvants commonly used in similar vaccines may be used but non-oil based adjuvants such as of the aluminium hydroxide type are preferred.
• the vaccine further comprises molecules derived from members of the Fasciola genus or other parasites. It is likely that other molecules, unrelated to GST, may also induce a protective immune response in ruminants and that a cocktail vaccine comprising these other molecules together with GST may be an effective vaccine.
• Figure 1 One dimensional SDS-PAGE analysis of the glutathione-binding molecules purified from a crude homogenate of F. hepatica adult worms by affinity chromatography on glutathione agarose. The position of the molecular weight markers is indicated (in kDa).
• FIG. 3 Comparison of the N-terminal sequences obtained for GSTs of F. hepatica (Fh) to the N-termini of GSTs of other helminths (Schistoceohalus solidus (Ss), Schistosoma mansoni (Sm), Schistosoma japonicum (Sj) and mammalian Mu class GSTs. Homologous regions are boxed. Rat (Rn), mouse (Mm); bovine (Bi) and human (Hs) GSTs are also represented. The bracketed residues indicate uncertain amino acid assignments.
• FIG. 4 Comparison of the sequence of tryptic and chymotryptic peptides of the GSTs of F. hepatica to homologous regions in GSTs of S. mansoni (Sm26), S. japonicum (Sj26) and the mouse (Mm GST1).
• CT18.3 chymotryptic peptide of F. hepatica: T0.7a, T0.7b, T16.3a, T16.3b, T16.2a, T16.2b: tryptic peptides of
• FIG. 6 EUSA analysis of native F. hepatica GST probed with antisera from sheep immunized with GST in Freund's adjuvant (A), infected with F. hepatica for 12 wks (•), infected with F. hepatica for 6 wks ( a ) and normal sheep serum (*).
• FIG. 7 Western blot analysis of native F. hepatica GST probed with antisera from different sheep.
• Panel A an amido black stain of the native protein
• panel B normal sheep serum
• panel C sera from sheep immunized with GST in Freund's adjuvant
• panel D sera from sheep infected with F. heoatica for 6 weeks
• panel E sera from sheep infected with F. hepatica for 12 weeks.
• Sera were used at a dilution of 1/100 (lane 1), 1/300 (lane 2) or 1/1000 flane 3). The position of the molecular weight markers is indicated (kDa).
• Panel A shows the average RBC hemoglobin levels over 36 weeks of infection with F. hepatica in uninfected control sheep ( ), infected control sheep
• Panel B shows average RBC hemoglobin levels in sheep over 36 weeks of infection in uninfected control sheep ( ), infected control sheep ( — ), GST group 1 vaccinated sheep
• Panel A shows the average aspartate aminotransferase serum levels over 36 weeks of infection with F. hepatica in serum from uninfected control sheep ( ), infected control sheep ( — ) and GST-vaccinated sheep ( ).
• Panel B shows average aspartate aminotransferase serum levels in sheep over
• Panel A shows the average L - gamma glutamyltransferase levels over 36 weeks of infection with F. hepatica in uninfected control sheep ( ), infected control sheep (- - -) and GST-vaccinated sheep ( ).
• Panel B shows average
• Panel A shows the average fecal egg counts over 36 weeks of infection with hepatica in infected control sheep (- - -) and GST-vaccinated sheep ( ).
• Figure 15 DNA sequence of the GST 7 cDNA.
• Figure 17. DNA sequence of the GST 47 cDNA.
• Figure 18. DNA sequence of the GST 50 cDNA.
• FIG. 19 Comparison of the amino acid sequences of cloned GST sequences and GST peptides of F. hepatica.
• Sm26 Mr 26,000 GST of S. mansoni: Sj26: Mr 26,000 GST of S. jaoonicumr Fh26a, Fh26b: N-terminal amino acid sequences of GSTs of F. hepatica;
• GST1 ,7,42,47,50 amino acid sequences predicted from the cloned GST cDNAs of F. hepatica. T.05, TO.7b/0.6, T21.5: tryptic peptides of F. hepatica: CT18.3:chymotryptic peptide of F. hepatica.
• the sequences have been aligned to maximise the homology. Dashes indicate unassigned residues.
• Fasciola hepatica adult worms used for purification of GSTs were collected from the livers of sheep slaughtered and processed at local abattoirs in Melbourne. The parasites were transported on ice, washed twice in phosphate buffered saline (PBS) and homogenized in TNET buffer (0.5% v/vTriton X-100 (Triton X-100 is a non-ionic detergent supplied by Rohm & Haas), 10mM EDTA, 0.15M NaCI, in 50mM Tris (pH 7.8) supplemented with 2mM phenyimethylsulphonyi fluoride) ata ratio of 1 ml /worm.
• PBS phosphate buffered saline
• TNET buffer 0.5% v/vTriton X-100
• Triton X-100 is a non-ionic detergent supplied by Rohm & Haas
• 10mM EDTA 0.15M NaCI
• 50mM Tris
• GST ⁇ soenzymes were purified by affinity binding to glutathione (GSH) agarose (Sigma, St Louis, USA). Briefly, TNET Iysates of adult worms were passed down a GSH agarose column, and the matrix washed with several volumes of PBS, prior to elution with a GSH containing buffer (1.5mg/ml GSH in 50mM Tris (pH 9.3) ) [7]. Fractions shown to contain protein were pooled, dialysed against PBS or distilled water and stored at -70°C. The GST content and purity were assessed by Coomassie blue and silver staining of SDS-PAGE gels. Generation of Peptides
• the chymotrypsin digest was prepared by addition of 200 ⁇ l 0.1 M NH 4 HC0 3 , pH 7.8, (C0 2 ) and 10/tg chymotrypsin (Worthington) and proteoiysis conducted at 37°C for 4 hours. Digestion was arrested by storage at -20°C.
• the void volume and peptide peaks suspect of heterogeneous content were refractionated on a Pep PRC 5/5 C 2 /C 18 reverse phase column (Pharmacia) most often using a 0-60% v/v AcN gradient in 0.1% v/v trifluoracetic acid. The elution was monitored at 214nm. Collected peptide peaks were stored at -20°C, and dried by vacuum centrifugation (Savant instruments, Hicksville, USA) prior to amino acid analysis.
• N-terminal and peptide sequencing was conducted at the Department of Veterinary Preclinical Sciences, University of Melbourne, using an ABI Model 471 A Protein Sequencer. Derivitized amino acids were resolved on a 25cm Zorbax PTH column (Dupont) (at 38°C) using isocratic delivery of the resolving buffer (5.529% v/v tetrahydrofuran, 30.17% v/v AcN,
• sample buffer (62.5 mM Tris-HCI containing 3% SDS, 50 mM dithiothreitol a ⁇ d 10% glycerol, pH 6.8), and electrophoresed under reducing conditions on 13% acrylamide slab gels [13].
• Relative molecular weights (M r ) were calculated with reference to protein molecular weight standards (Biorad, Richmond, USA).
• Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose paper was performed according to the method of Bumette [16], with a transfer time of 18 hours at 15 volts.
• the nitrocellulose sheet was blocked with 5 % skim milk powder in PBS for 3 hours at room temperature.
• the antiserum was diluted 1 in 100 in PBS, added to the nitrocellulose sheet, and incubated for 1 hour.
• the sheet was washed three times in PBS containing 0.1 % Tween 20.
• Affinity-purified rabbit anti-sheep immunogiobulin (Biorad) or goat anti-rabbit immunoglobulin (Kinkegaard and Perry Labs, Gaithersburg, USA) was diluted 1 in 300 in PBS and added to the sheet and incubated at room temperature for 1 hour.
• the sheet was washed 3 times in PBS / 0.1 % Tween 20 (Tween 20 is a non-ionic detergent) and developed using 4 ml of a 3 mg/ml solution of 4-chloro-1-napthol (Sigma) in cold methanol mixed with 20 ml PBS containing 12 ⁇ l of hydrogen peroxide.
• the location of the transferred protein was established by staining in a solution of 0.004 % amido black in 45 % methanol / 10 % acetic acid. lodination of proteins
• the native GSTs of F. hepatica were radioiodinated using the Boiton and Hunter procedure [17].
• Polyvinyl microtitre plates were coated overnight at 4-C with 50 ⁇ l purified GST (5 ⁇ g/ml) in 0.1 M sodium carbonate buffer (pH 9.5). Sera were diluted in EUSA buffer (0.1 M Tris HO, 0.5 M NaCI, 2 mM EDTA, 0.05 % Tween 20, 0.05 mM thiomersal, pH 8.0) supplemented with 0.2 % bovine serum albumin, and 50 ⁇ l of the appropriate dilution was incubated in the microtiter plate for 1 hour at 37° C. The wells were washed 3 times between incubations with PBS containing 0.1% v/v Tween 20.
• Affinity-purified rabbit anti-sheep immunoglobulin conjugated to horse radish peroxidase was diluted in EUSA buffer, 50 ⁇ l was added to each well and incubated for 1 hour at 37° C.
• the substrate 1 mM 2,2-Azinobis (3-ethylbenzthiazole sulphonic acid) (ABTS) in 0.062 M citric acid / 0.076 M Na 2 HP0 4 pH 4.0, 0.03 % hydrogen peroxide, was added to each well. After 1 hour, the optical density at 414 nm was measured with an automated Titertek Multiskan spectrophotometer.
• a group of 10 sheep were immunized by subcutaneous injection of 10Q ⁇ g of purified GST of F. hepatica in Freund's complete adjuvant (FCA) 16 weeks prior to infection followed by a boost with 100 ⁇ g of purified GST in Freund's incomplete adjuvant (IFA) 12 weeks prior to infection.
• FCA hepatica in Freund's complete adjuvant
• IFA Freund's incomplete adjuvant
• the sheep were given subsequent boosts of 100 ⁇ g of purified GST in PBS at approximatly 4 week intervals throughout the 52 weeks of the trial.
• a group of 10 control sheep were treated identically, with PBS substituted for the GST antigen.
• a group of 8 sheep were not immunized.
• Serum taken at each time interval was assayed for the liver enzymes aspartate aminotransferase (EC 2.6.1.1.) (AST) [19] and L-gamma glutamyltransferase (EC 2.3.2.2.) (GGT) [20] and red blood cell (RBC) hemoglobin [21] on a Roche Cobas MIRA automatic analyser (Basel, Switzerland). Serum was stored frozen at -20*C until use.
• AST aspartate aminotransferase
• GTT L-gamma glutamyltransferase
• RBC red blood cell
• Fecal egg counts were performed by the method of Kelly et al [22] with the following changes.
• One gram of feces was suspended in 9ml of water and passed through a sieve into a tapered urine flask to remove coarse fecal material.
• the eggs were allowed to settle for 6 minutes and most of the supematent removed. This procedure was repeated once and yielded about 10 ml of sediment containing F. hepatica eggs.
• Several drops of 0.1 % new methylene blue were added to the final sediment to a volume of 10 ml and poured into a square lined petri dish. The number of eggs/g feces were counted under a dissecting microscope.
• RNA was extracted from adult worms of the Compton strain of F. hepatica by the method of Chirgwin et al [24]. Poly(A) + RNA was selected by oligo dT chromatography [25]. The cDNA libraries were constructed in phage vectors gtl 1 a ⁇ dfcAP by CLONTECH (Palo Alto, USA) using the procedure of Gubler and Hoffman [26].
• the cDNA libraries were screened for expression of GSTs of F. hepatica using the Protoblot method as described in the Protoblot Technical Manual purchased from PROMEGA (Madison, USA). The library was screened with a rabbit antiserum raised to the purified GSTs of F. hepatica at a dilution of 1/600. Filters were blocked in a buffer containing
• Antibodies in the rabbit anti-GST serum were depleted of specificities to sequences expressed in the GST1 clone before the ⁇ ZAP library was rescreened to identify other GSTs of F. hepatica.
• Undiluted rabbit antiserum (1ml) was incubated with 1ml of a sonicate of E. coli expressing ⁇ -galactosidase for 16 hours at 4°C to deplete anti- E. coli specificities.
• This depleted serum was diluted to 1/600 with PBS and 10m! of this serum was incubated on a filter to which an induced confluent lawn of clone GST1 had been absorbed. After 1 hour at room temperature, the serum was removed and used to screen the/ZAP library.
• One positive plaque was obtained (termed GST 7) which was rescreened to purity.
• Radiolabelled probes were prepared as described by using the BRL (Gaithersburg, USA) nick translation kit as recommended by the supplier.
• Phagemid DNA containing cDNA inserts from positive ⁇ ZAP phage clones was isolated by excision in vivo of the pBluescript phagemid under the conditions recommended by Stratagene (La Jolla, USA). Phagemid DNA was extracted by the method of Birnboim and Doly [27]. Double-stranded DNA sequencing of cDNA inserts was performed by the chain termination method [28].
• the GST fraction comprises protein extracted from a population of individual adult worms isolated from several infected sheep livers. Since each sheep could be infected with several strains of F. hepatica which may exhibit sequence polymorphisms within GST isoenzymes, the multiple protein components observed within our GST fraction could represent allelic variants of one or a few GST isoenzymes within the polymorphic fluke population studied. Alternatively, each component could be the product of an individual GST gene within a clonal fluke population. Amino acid sequence of native GSTs of F. hepatica
• N terminal amino acid sequences of the purified F. hepatica GSTs revealed two different but related sequences. Comparison of these sequences (Fh26a, Fh26b) with the corresponding regions of Schistosoma [7,30,31] and known mammalian GSTs [31,37] showed very high levels of homology (Fig. 3). Conservation of several key regions of sequence resulted in identities of 52-76% and 55-77% for Fh26a and Fh26b respectively (Table 1).
• T21.5b and T21.6a are identical and show 69% identity with the Oterminal region of the M r 26,000 GST of S. japonicum and S. mansoni.
• the RBC hemoglobin levels in the uninfected control animals remained consistently high around a mean of 12 g/L over the period of the trial.
• the GST vaccinated sheep displayed levels consistently orientated around the median between the uninfected and the infected control animals (Fig 8a).
• Fig 8b When the GST vaccinated animals were analysed further as two sub-populations (Fig 8b), based solely upon relative RBC haemoglobin levels through the trial, it was found that 4 of the animals (GST group 1) displayed consistently higher levels of RBC hemoglobin than the infected controls, while the remaining 5 animals (GST group 2) demonstrated a decrease with time, consistent with the infected controls.
• AST serum levels were analysed to assess the level of liver parenchymal damage in the trial animals.
• the GST-vaccinated animals consistently displayed levels of serum AST similar to the infected control animals (Fig 9a).
• the GST-vaccinated animals were assessed as 2 sub-populations (Fig 9b)
• the GST group 1 animals displayed lower serum levels over the initial 10 weeks with a slightly delayed maximum reached at week 6 compared to week 4 in the infected control animals.
• the animals in GST group 2 did not display any differences in AST serum levels from the infected control animals.
• GGT levels in serum are an indicator of damage to the liver and specifically the bile ducts and were analysed to monitor damage resulting from the establishment of parasites in the bile ducts.
• the level of GGT in the GST-vaccinated animals demonstrated a profile similar to that recorded for the infected control animals (Fig 10a) with a rise in the levels of enzyme in serum detectable by week 2, peak values by week 12 and a slow decrease after this time. No comparable release of GGT into serum was detected in the uninfected control animals.
• GST-vaccinated animals were analysed as sub-populations (Fig 10b)
• GST group 1 displayed lower GGT levels over the initial 12 weeks and with maximal levels not attained until week 14.
• GGT levels in the GST group 2 again coincided with the infected controls. This suggests that the GST group 1 subpopulation of animals have a decreased and delayed onset of liver damage compared with the controls and the GST group 2 subpopulation.
• the sheep were slaughtered over a period of 13 weeks (weeks 44 - wk 57), post infection, and the worm burdens within each liver were ascertained ( Figure 12 and Table 2).
• the 10 infected controls sacrificed to date, contained an average of 241 parasites in comparison to the GST-vaccinated animals with a mean of 107 parasites representing an overall reduction in worm burden of 55 % (p ⁇ 0.001).
• the GST group 1 group exhibited a mean worm count of 54, representing a reduction of 77% (p ⁇ 0.001)
• the GST group 2 group exhibited a mean worm count of 149, representing a reduction of 38% (p ⁇ 0.025).
• one third of the GST-vaccinated animals exhibited worm burdens of less than 15 % of the mean burden in the control animals.
• Rabbit antiserum was raised to the purified GST fraction by subcutaneous injection of F. hepatica GST in Freund's adjuvant. This antiserum identifies various GST species of M p 26,000 and 26,500 on Western blots of the purified GST fraction separated by two dimensional SDS-PAGE (Fig 13). This antiserum was used to isolate cDNA sequences of
• GST1 and GST7 Two cDNA clones (termed GST1 and GST7) were identified.
• the cDNA sequence of GST7 was used to isolate other homologous cDNA sequences in the library by DNA-DNA hybridization which identified 3 other cDNA sequences (termed GST42, GST47 and GST50).
• the amino acid sequences predicted by each of the cDNA sequences is shown in Fig 19 together with an alignment with the M r 26,000 GST sequences of Schistosoma [7,30].
• Each cDNA sequence predicts a single open reading frame.
• the GST 1 amino acid sequence begins 22 amino acids from the N terminus of GST peptides (Fh26b) and shows a degree of homology with this sequence.
• the GST 7 amino acid sequence begins 7 amino acids from the N terminus of GST (Fh26a) and is identical to this sequence.
• the GST47 amino acid sequence begins 6 amino acids from the N terminus of GST (Fh 26a, b) and shows high homology with these sequences.
• the GST42 and GST50 sequences are much shorter.
• the GSTs of adult worms of F. hepatica comprise two major components of approximate M 26,000 and 26,500 which can be further fractionated into 10-11 components by two dimensional SDS-PAGE.
• Direct sequencing of the GST fraction of F. hepatica identified two major N-terminal sequences.
• peptides derived from internal or C-termi ⁇ al regions of GSTs were identified by homology with other known GSTs. From these data, it is evident that the glutathione binding molecules purified do represent the GSTs of hepatica.
• the isolation and sequencing of near identical peptides indicates the high degree of heterogeneity in the F. hepatica GST fraction and implies the expression of multiple GST genes in this parasite.
• the health of the animals and the progression of the infection was monitored by the assay of several biochemical parameters in erythrocytes and serum.
• a subpopulation of the GST vaccinated animals (GST group 1) displayed a clear biochemical pattern consistent with both a reduced worm burden as well as a delay in the establishment of these worms in the bile ducts.
• the subsequent finding of a 77 % reduction (p ⁇ 0.001 ) in worm burden in these animals was complementary to the biochemical findings.
• a statistically significant reduction (p ⁇ 0.025) in worm burden of 38 % was also demonstrated in the GST group 2.
• An overall reduction in worm burden of 55 % (p ⁇ 0.001) was demonstrated in the vaccinated group as a whole.
• the nature of the protective immune response directed against the parasite remains uncertain.
• a strong humoral response to GST of F. hepatica has been induced in all the vaccinated animals but the members of GST group 1 do not exhibit a differentially higher antibody titre relative to the GST group 2 animals. It is therefore uncertain if a humoral response and/or a T-cell response is necessary to induce the protective effect observed.
• the target of the immune attack could be GST in the metacercariae and/or in the newly excysted juvenile resulting in the subsequent damage and elimination of the parasite.lt is also possible that immune attack on the GST of F. hepatica has facilitated induction of a response to a novel parasite antigen leading to the death of the parasite.

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• 1990
  • 1990-01-31 WO PCT/AU1990/000027 patent/WO1990008819A1/en not_active Application Discontinuation
  • 1990-01-31 JP JP2502498A patent/JPH04507235A/ja active Pending
  • 1990-01-31 BR BR909007070A patent/BR9007070A/pt not_active Application Discontinuation
  • 1990-01-31 AU AU50283/90A patent/AU634754B2/en not_active Ceased
  • 1990-01-31 NZ NZ232327A patent/NZ232327A/en unknown
  • 1990-01-31 CA CA002045663A patent/CA2045663A1/en not_active Abandoned
  • 1990-01-31 EP EP19900902123 patent/EP0456662A4/en not_active Ceased
• 1991
  • 1991-07-29 NO NO91912942A patent/NO912942L/no unknown

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